Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis, remains a major cause of human morbidity and mortality, particularly in the developing world. Chronic M. tuberculosis infection requires long- term interactions between the bacterium and host immune system, and tissue macrophages play key roles in the outcome of infection. Although technologies to monitor global changes in host gene expression have catalyzed our understanding of the important roles for TLR activation and interferon during TB infection, modulation of transcription represents only one of many cellular responses to bacterial infection. Post- translational modification of proteins, such as ubiquitylation, phosphorylation, and acetylation play a role in regulating virtually every cellular process. How these signaling events lead to observed changes in metabolic pathways, autophagy, and vesicular trafficking during bacterial infection remain unknown. Furthermore, how these processes may be manipulated by pathogens is crucial for understanding the pathogenic strategies of microorganisms. This proposal seeks to use powerful new proteomic technologies to globally quantify changes in ubiquitylation in order to identify novel functional macrophage responses to M. tuberculosis infection. Our preliminary experiments utilizing this approach have uncovered profound changes in host protein ubiquitylation in response to intracellular pathogens. These studies have provided the first glimpse into a vast unknown of post-translational modifications during innate immune responses. Our hypothesis is that these changes play fundamental roles in shaping the subsequent innate responses to infection by controlling autophagy, metabolism, protein degradation, and signaling, and may be manipulated by pathogens for their own benefit. Our preliminary genetic work indicates that novel pathways are controlled by ubiquitylation during infection and are capable of restricting M. tuberculosis growth in macrophages, further validating the power of this approach to uncover new biology. Ultimately, our long-term goal is to harness these powerful immune mechanisms for therapeutic purposes.